alignment-nf

Nextflow pipeline to perform BAM realignment or fastq alignment, with/without local indel realignment and base quality score recalibration.

Overview of pipeline workflow

Prerequisites

For the basic fastq files alignment without indel realignment, base quality score recalibration, nor alternative contif handling, the script requires the following programs:

• nextflow. Install with

   curl -fsSL get.nextflow.io | bash

  And move it to a location in your $PATH (e.g., /usr/local/bin):

   sudo mv nextflow /usr/local/bin

• bwa
• samblaster
• sambamba and the following files:
• a fasta reference genome with its index files (.fai, .sa, .bwt, .ann, .amb, .pac; in the same directory)
• a folder with fastq files

In addition, for the bam files realignment:

• samtools
• a folder with bam files

For the adapter sequence trimming:

• AdapterRemoval

For the ALT contigs handling, additional softwares and scripts are required:

• the k8 javascript execution shell (e.g., available in the bwakit archive)
• javascript bwa-postalt.js and the additional fasta reference .alt file from bwakit must be in the same directory as the reference genome file.

For the indel realignment:

• GATK GenomeAnalysisTK.jar
• GATK bundle VCF files with lists of indels (recommended: 1000 genomes and Mills gold standard VCFs)

For the base quality score recalibration:

• GATK GenomeAnalysisTK.jar
• GATK bundle VCF files with lists of indels and SNVs (recommended: 1000 genomes indels, Mills gold standard indels VCFs, dbsnp VCF)
• bed file with intervals to be considered

Usage

nextflow run iarcbioinfo/alignment-nf --input_folder input --fasta_ref hg19.fasta --out_folder output

Enable adapter trimming

To use the adapter trimming step, you must add the --trim option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --input_folder input --fasta_ref reference/hs38DH.fa -out_folder output --trim

Enable ALT mode

To use the alternative contigs handling mode, you must provide the path to an ALT aware genome reference (e.g., hg38) AND add the --alt option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --input_folder input --fasta_ref reference/hs38DH.fa --js /user/bin/k8/k8 --postaltjs /user/bin/bwa-0.7.15/bwakit/bwa-postalt.js -out_folder output --alt

Enable local indel realignment

To use the local indel realignment step, you must provide the path to the GATK jar file, the GATK bundle folder, AND add the --indel_realignment option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --GATK_bundle GATKbundle/hg19 --input_folder input --fasta_ref reference/hg19.fa --GATK_folder /user/bin7GATK-3.6-0 --out_folder output --indel_realignment

Enable base quality score recalibration

To use the base quality score recalibration step, you must provide the path to the GATK jar file, the GATK bundle folder, a bed file, AND add the --recalibration option, as well as satisfy the requirements above mentionned. For example:

nextflow run iarcbioinfo/alignment-nf --GATK_bundle GATKbundle/hg19 --input_folder input --fasta_ref reference/hg19.fa --GATK_folder /user/bin7GATK-3.6-0 --intervals reference/hg19_intervals.bed --out_folder output --recalibration

All parameters

PARAMETER	DEFAULT	DESCRIPTION
--help	null	print usage and optional parameters
--input_folder	.	input folder
--fasta_ref	hg19.fasta	genome reference
--cpu	8	number of CPUs
--mem	32	memory
--mem_sambamba	1	memory for software sambamba
--RG	PL:ILLUMINA	sequencing information for aligned (for bwa)
--fastq_ext	fastq.gz	extension of fastq files
--suffix1	_1	suffix for second element of read files pair
--suffix2	_2	suffix for second element of read files pair
--out_folder	.	output folder for aligned BAMs
--intervals		bed file with interval list
--GATK_bundle	bundle	path to GATK bundle files
--GATK_folder	.	path to GATK GenomeAnalysisTK.jar file
--trim	false	enable adapter sequence trimming
--indel_realignment	false	perform local indel realignment (GATK)
--recalibration	false	perform quality score recalibration (GATK)
--js	k8	path to javascript interpreter k8
--postaltjs	bwa-postalt.js"	path to postalignment javascript bwa-postalt.js
--alt	false	enable alternative contig handling (for reference genome hg38)